We have further developed our project to assess HER2 overexpression in breast carcinoma xenografts (mouse model), using near infrared fluorescence imaging. Novel specific probes, based on small Affibody molecules, in combination with in-house instrumentation, allowed us to characterize tumors with different levels of HER2 overexpression in the cancer cells in vivo. Two fluorescent dyes (AlexaFluor 750 and DyLight) with different binding/washout properties were tested. It was shown that HER2 overexpression can be estimated from analysis of a series of the tumor fluorescence images, if the probe pharmacokinetics is taken into account by a ligand-receptor model. Good correlation between results of our in vivo method and conventional ex vivo techniques has been observed. We were able to monitor changes in the tumor due to its treatment with a known anti-cancer drug 17-DMAG in the mouse model. Lifetime is another characteristic of fluorescence that can be estimated from time-resolved imaging, performed with our instrumentation. With a proper dye it can potentially provide information about fluorophore environment ((pH, temperature, tissue oxygen content, nutrient supply, and bioenergetic status)) that can be of real diagnostic value. In collaboration with Dr. Achilefu from Washington University we have recently developed a novel pH-sensitive dye and performed its initial tests in the phantoms. We are also investigating changes in the fluorescence lifetime for HER2 specific probes in the tumor in vivo (mouse model) in order to assess the properties of the cancer cells that maybe important to optimize treatment. The two-photon imaging project has continued as a study on an in-vivo device. The device is only able to collect epifluorescence and does so using a collection dish at some distance from the objective. It was important to study the effects of the tissue properties of the lens and the distance of the collector from the illumination center. These two factors are crucial in choosing the configuration of the device. It transpired from simulations that the key issue was distance and we could determine a distance for the reflection collector to be set at from the illumination location to ensure that the total epi-collection occurred. Polarization imaging is a promising tool to visualize hidden structures below the tissue surface. Analysis of these structures, for example the collagen network, can be used to assess the possible transition from normal tissue function to diseased tissue. We have developed sophisticated techniques of data analysis, based on Pearson correlation procedure, that allow to enhance the image quality and reveal regions of high statistical similarities within the noisy images, making possible characterization of subsurface structural features of biological tissues. To realize the potential of the method we have designed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light. Preliminary experiments have demonstrated that designed polarization adapter in combination with developed data analysis algorithms can provide quantitative information on collagen structure. We have incorporated this adapter into a conventional colposcope and started a clinical protocol to study tissue structures in the cervix. Data on 10 healthy volunteers have been collected at luteal and follicular phases of the cycle, and are being analyzed to optimize the system and update the clinical protocol The oncology community is testing a number of novel targeted approaches for use against a variety of cancers. With regard to monitoring vasculature, it is desirable to develop and assess noninvasive and quantitative techniques that can not only monitor structural changes, but can also assess the functional characteristics or the metabolic status of the tumor. We are testing three potential noninvasive imaging techniques to monitor patients undergoing an experimental therapy: infrared thermal imaging (thermography), laser Doppler imaging (LDI) and multi-spectral imaging. These imaging techniques are being tested on subjects with Kaposi s sarcoma (KS), a highly vascular tumor that occurs frequently among people infected with acquired immunodeficiency syndrome (AIDS). Cutaneous KS lesions are easily accessible for noninvasive techniques that involve imaging of tumor vasculature, and they thus represent a tumor model in which to assess certain parameters of angiogenesis. The KS studies are ongoing clinical trials under four different NCI protocols. We have shown that our multi-modality techniques can non-invasively monitor the functional properties of the tumor and surrounding tissues and has the potential to predict treatment outcomes. Recent work has been performed on obtaining these angiogenic markers (blood volume and oxygenation) in real time. Using novel data analysis tools based on Principal Component Analysis, we have shown that a KS lesion can be imaged and assessed in means of the functional state of the tumor in real time. In combination with OCT, we have recently shown quantitative results of blood volume and oxygenation, which were obtained in real time. We have also found that quantitative results can be obtained if the underlying skin structures are being taken into account. In order to obtain information about these structures, we have developed a spectral domain Optical Coherence Tomography (OCT) system, which gives 3D images of the skin.